Electrical perception of the 'death message' in Chara: analysis of K+-sensitive depolarization

Wounding electrical responses were studied in Chara corallina. Specimens comprising two adjoining internodal cells were prepared. When one cell (victim cell) was cut, the other cell (receptor cell) generated four kinds of depolarization: (i) rapid depolarization; (ii) long-lasting depolarization; (iii) action potentials; and (iv) small spikes. In the present study, attention was focused on the long-lasting depolarization. A decrease in the electrical resistance suggested activation of ion channel(s). The duration of the depolarization was sensitive to the external ions. K(+) significantly prolonged the depolarization. On the other hand, Ca(2+), Mg(2+) and Na(+) had a tendency to shorten the duration prolonged by K(+). When a nodal end was continuously flushed with a medium lacking K(+), the depolarization was significantly shortened. Treatment of the nodal end with artificial cell sap for 2 min induced a long-lasting depolarization similar to that induced by cutting the victim cell. These findings suggested the involvement of K(+) released from the victim cell in generating the long-lasting depolarization by the receptor cell.     

Electrical perception of the 'death message' in Chara: characterization of K+ -induced depolarization

When the nodal end of an internodal cell of Chara corallina was subjected to a pulse treatment with artificial cell sap, a depolarization lasted in artificial pond water. This depolarization could also be induced by pulse treatment with KCl solution, indicating that K+ in the artificial cell sap is responsible (K+ -induced depolarization). The depolarization was prolonged in the presence of 2 mM KCl and the prolonged depolarization was terminated by supplementing with either CaCl2, MgCl2 or NaCl. These results supported the hypothesis that K+ released from the killed cell is responsible for generation of the wound-induced membrane depolarization.     

Possible link of different slow calcium signals generated by membrane potential and hormones to differential gene expression in cultured muscle cells

The study of fluorescent calcium signals from cultured rat myotubes has provided interesting results in the past few years. Both K+ depolarization and tetanic electrical stimulation were shown to produce slow Ca2+ signals, unrelated to contraction and associated to regulation of gene expression in cultured rat myotubes. We studied the effect of IGF-I, insulin and testosterone on intracellular Ca2+ in cultured muscle cells. Insulin produced a fast (< 1 s) and transient [Ca2+] increase lasting less than 10 s. IGF-I induced a transient [Ca2+] increase, reaching a fluorescence peak 6 s after stimulus, to return to basal values after 60 s. Testosterone induced delayed (35 s) and long lasting (100-200 s) signals, frequently associated with oscillations. IGF-I, testosterone and electrical stimulation-induced Ca2+ signals were shown to be dependent on IP3 production. All of these Ca2+ signals were blocked by inhibitors of the IP3 pathway. On the other hand, insulin-induced Ca2+ increase was dependent on ryanodine receptors and blocked by either nifedipine or ryanodine. The different intracellular Ca2+ patterns produced by electrical stimulation, testosterone, IGF-I and insulin, may help to understand the role of intracellular calcium kinetics in the regulation of gene expression by various stimuli in skeletal muscle cells.     

Amplification of calcium signals at dendritic spines provides a method for CNS quantal analysis

It has been proposed that the small volume of a dendritic spine can amplify Ca2+ signals during synaptic transmission. Accordingly, we have performed calculations to determine whether the activation of N-methyl-D-aspartate (NMDA) type glutamate receptors during synaptic transmission results in significant elevation in intracellular Ca2+ levels, permitting optical detection of synaptic signals within a single spine. Simple calculations suggest that the opening of even a single NMDA receptor would result in the influx of approximately 310 000 Ca2+ ions into the small volume of a spine, producing changes in Ca2+ levels that are readily detectable using high affinity Ca2+ indicators such as fura-2 or fluo-3. Using fluorescent Ca2+ indicators, we have imaged local Ca2+ transients mediated by NMDA receptors in spines and dendritic shafts attributed to spontaneous miniature synaptic activity. Detailed analysis of these quantal events suggests that the current triggering these transients is attributed to the activation of <10 NMDA receptors. The frequency of these miniature synaptic Ca2+ transients is not randomly distributed across synapses, as some synapses can display a >10-fold higher frequency of transients than others. As expected for events mediated by NMDA receptors, miniature synaptic Ca2+ transients were suppressed by extracellular Mg2+ at negative membrane potentials; however, the Mg2+ block could be removed by depolarization.     

Intracellular axial current in Chara corallina reflects the altered kinetics of ions in cytoplasm under the influence of light

Recent experiments demonstrate that the concentration of Ca2+ in cytoplasm of Chara corallina internodal cells plays important role in electrical excitation of the plasma membrane. The concentration of free Ca2+ in the cytoplasm -[Ca2+]c is also sensitive to visible light. Both phenomena were simultaneously studied by noninvasive measuring action potential (AP) and magnetic field with a superconducting quantum interference device magnetometer in very close vicinity of electrically excited internodal C. corallina cells. A temporal shift in the depolarization maximum, which progressively occurred after transferring cells from the dark into the light, can be explained by the extended Othmer model. Assuming that the change in membrane voltage during the depolarization part of AP is the direct consequence of an activation of [Ca2+]c sensitive Cl- channels, the model simulations compare well with the experimental data. We can say that we have an example of electrically elicited AP that is of biochemical nature. Electric and magnetic measurements are in good agreement.     

Electrical perception of "death message" in Chara: involvement of turgor pressure

Plants show various defense responses upon wounding. Surviving cells must perceive a "death message" from killed cells in order to start the signal processing that results in defense responses. The initial step in perception of the death message by a surviving cell was studied by taking advantage of the filamentous morphology of characean algae. A specimen comprising two adjoining internodal cells was prepared. One cell (the victim cell) was killed by cutting and any changes in the membrane potential of the neighboring cell (the receptor cell) were analyzed. Upon cutting the victim cell, at least one of three kinds of response were induced in the receptor cell: (1) slow depolarization lasting more than 10 min, (2) action potentials and (3) small spikes. The first of these response types, slow depolarization, was ubiquitous and is the focus of the present study. Two cell properties were essential for generation of this depolarization. (1) Presence of high cell turgor pressure was necessary. (2) The depolarization was generated only at the nodal end of the receptor cell, not at the flank. I concluded that the death message from the killed cell contains the information that turgor pressure has been lost. The mechanism by which this is translated into the slow depolarization of the receptor cell was discussed.     

乙酰胆碱对黄化玉米幼苗中胚轴通透性与物质运转的调控效应

对乙酰胆碱处理前后的黄化玉米 (ZeamaysL .)幼苗中胚轴切段节和节间的通透性和物质运转进行了比较。用电阻法测定通透性 ;通过加在胚芽鞘切口的经共质体转运的特异荧光物质羧基荧光素 (carboxyfluorescein ,CF)的分布检察物质运转。节间由柱状的、排列方向多少和长轴方向平行的细胞构成 ;而构成节的细胞短、排列不规则。结果表明跨节比电阻比节间的高 ,通透性低的短的节细胞比柱状节间细胞累积更多的CF。 0 .1mmol/L乙酰胆碱 (ACh)处理增加了节和节间的通透性 ,促进了CF的运转。物质在节和节间运转的不同 ,以及ACh对此的调控效应很大程度上可归结于细胞通透性的差异     

Electrotonic Coupling between Adjacent Internodal Cells of Chara braunii: Transmission of Action Potentials beyond the Node

Many nodal cells are interposed between two internodal cellsof Chara braunii. When an action potential conducted in an internodereached the node, no electrical activation in the nodal cellscould be found, although an area of the membrane bordering thenodal cells in this internode was partially activated (end-membraneaction potential). When the action potential approached thenode along the stimulated internode, an electrotonic potentialchange (depolarization) was produced in the other internode.This depolarization was greatly depressed by the end-membraneaction potential of the stimulated internode, so that hardlyany transmission took place. The ratio of the potential changein the surface membrane of the adjoining ("postsynaptic") internode(cell b) to that of the stimulated one (cell a), the couplingratio, e_b/e_a, can be estimated from a simple equivalent circuitof the nodal region composed of two surface-membrane resistances(R_a, R_b) and intercellular resistance (R_n). If R_n remains thesame, a higher ratio should be produced with a larger R_b, butthe ratio does not depend on any change in R_a, which could beproved experimentally. Transmission of the action potentialbeyond the node was frequent when the coupling ratio was increasedand when the threshold that elicits the action potential waslowered by immersing the node in a K or Na salt solution. ¹ Present address: Department of Physiology, Tohoku UniversitySchool of Dentistry, Sendai 980, Japan. (Received December 1, 1980; Accepted January 23, 1981)     

Modes of Mechanical Failure of Hollow, Septate Stems

Three general modes of mechanical failure were observed whenhollow, septate stem segments ofArundinaria técta(Poaceae)were axially compressed and caused to fail: (1) rupture of tissuesat the opposing ends of the nodal transverse diaphragm parallelto the plane of stem flexure; (2) localized catastrophic transverseinvagination of internodes (Brazier buckling); and (3) longitudinalrupture of internodal walls along the convex surface of stemflexure attended by nodal tissue shearing. The frequencies ofoccurrence of these three modes were not equivalent among the100 stem segments examined; stem failure was dominated by nodaltissue shearing (i.e. 67% of the nodes failed by shearing; 52%of the internodes longitudinally invaginated; 27% of the internodesfailed in Brazier buckling; 21% of the internodes failed byrupturing). Computer simulations of axially compressed stemsegments consisting of one node and one internode composed ofnon-linearly elastic, anisotropic materials (tissues) successfullypredicted observed strain patterns and revealed that the pathwayto stem failure contains a bifurcation point below which deformationpatterns coalesce on a single configuration, and above whichstem failure by Brazier buckling or nodal tissue shearing areessentially mutually exclusive responses to excessive stem flexure.The patterns of actual and simulated stem flexure were consistentwith the hypothesis that nodes store strain energy as stemsflex, and release this energy to restore stem shape when bendingforces abate (i.e. nodes operate as spring-like joints). However,strain energy ‘sinks’ identified by simulationswere also located in bending internodal walls which can storeelastic strain energy to do work. Nodal diaphragms and internodalwalls likely comprise a complex and global rather than a simpleand local elastic system for the recovery of stem shape.Copyright1998 Annals of Botany Company Stem flexure; mechanical failure; stem nodes; internodes; plants; biomechanics; finite element analyses; Brazier buckling.     

Intercellular Transport of Macromolecules in Nitella

We injected three different fluorescein isothiocyanate (FITC)-labeledproteins, two different FITC-labeled dextrans and the photoproteinaequorin (the molecular weight of each being more than 20 kDa)into internodal cells of Nitella. All macromolecules with molecularweights equal to or less than 45 kDa moved from the injectedcell to the neighboring nodal and internodal cells within 24h after injection. The injected aequorin emitted light in theadjacent internodal cell upon a transient increase in the cytoplasmicconcentration of Ca²⁺, an indication that the aequorin retainedits function after transport between cells. (Received November 2, 1991; Accepted March 7, 1992)     

Influence of Cytoplasmic Streaming and Turgor Pressure Gradient on the Transnodal Transport of Rubidium and Electrical Conductance in Chara corallina

In order to study the transnodal transport of Rb⁺ in internodalcells of Chara corallina, a low-temperature loading system wasestablished to separate the loading process from the transportprocess. Tandem cells, consisting of internode-node-internode,were isolated from algal plants. Treatment of a single internodewith 100 mM RbCl at 5°C for 30 min caused an accumulationof 43 mM Rb⁺ in the cytoplasm of this cell (= source cell),but no Rb⁺ was found in the other internode (= sink cell) ofthe tandem cells. In 40 min after a return to 25°C, about12% of the Rb⁺ loaded in the source cell was transported intothe sink cell. The apparent transnodal permeability of Rb⁺ wascalculated to be 4.6 x 10^–7 m.s^–1. Under the assumptionthat the total cross-sectional area of plasmodesmata occupies10% of the nodal area, the diffusion coefficient of RbCl throughplasmodesmata was calculated to be 2.3 x 10^–11 m².s^–1which is about 1% of the free diffusion coefficient in water(2 x 10^–9m².s^–1). The transnodal transport of Rb⁺ was intimately correlated withthe rate of cytoplasmic streaming. The rate of streaming inboth the source and sink cells was varied either by treatingthe cells with cytochalasin B (CB) or by lowering the temperature.The transport rate correlated with the streaming rate irrespectiveof the method used. Since the level of ATP was not influencedby CB or low temperature, the transnodal transport is assumedto be the result of passive diffusion process through plasmodesmata. A turgor pressure gradient across the node decreased both thenodal electrical conductance and the transnodal transport ofRb⁺. By contrast, the exposure of both internodal cells to asolution of sorbitol had no effect on either of them. A turgorpressure gradient of 240 mOsm decreased the transport of Rb⁺in the first hour to 3% of the control, while it decreased thenodal conductance to about 50%. The increase in the electricalresistance occurred on the junction side between the node andthe internode that was treated with sorbitol. Cytochalasin Ehad no effect on the nodal electrical resistance. It is assumedthat plasmodesmata are equipped with a valve-like mechanismwhich is sensitive to the gradient of turgor pressure acrossthe node and is not regulated by an actomyosin system. (Received February 15, 1989; Accepted April 20, 1989)     

Membrane depolarization induced by transcellular osmosis in internodal cells ofNitella flexilis

Summary Membrane depolarization induced by transcellular osmosis was studied using internodal cells ofNitella flexilis. Transcellular osmosis was induced by using sorbitol or methanol as the osmotic agent. In the endosmotic cell half, the membrane often generated an action potential and depolarized further with a concomitant decrease in membrane resistance. This osmosis-induced depolarization was a graded response dependent on the external osmotic gradients. However, in the exosmotic cell half, both membrane potential and membrane resistance changed insignificantly. Membrane depolarization occurred also in cells made inexcitable by bathing in 0.1–1 mM KCl solution.Effects of temperature and internal osmotic pressure on osmosis-induced depolarization were investigated. The magnitude of depolarization at low temperature (2 or 4°C) was larger than that at room temperature (around 20°C). Membrane depolarization was accelerated by lowering the internal osmotic pressure and inhibited by raising it.Not only the plasmalemma but the tonoplast also responded significantly to endosmosis.     

Immunolocalisation of the cytoskeleton to plasmodesmata of Chara corallina

The macromolecular structure of plasmodesmata in the giant celled freshwater alga, Chara corallina, was examined using antibodies against cytoskeletal elements. The large internodal cells of Chara are separated by a nodal complex of smaller cells which are interconnected by plasmodesmata. Putative plasmodesmata-associated proteins can be identified by a comparison of proteins extracted from preparations of clean walls of nodal complexes and those extracted from the external walls of internodal cells which have no plasmodesmata. Actin and tubulin were identified in the protein extracts of nodal walls and the cytoplasm of nodes and internodes but not in the extracts of internodal external walls. Immunogold labelling confirmed the localisation of actin and myosin to plasmodesmata of Chara.     

Involvement of protein synthesis in recovery from refractory period of electrical depolarization induced by osmotic stimulation in <Emphasis Type="Italic">Chara corallina</Emphasis>

Upon addition of sorbitol to the external medium of an internodal cell of Chara corallina, a transient depolarization is induced at its nodal end (Shimmen in Plant Cell Physiol 44:1215–1224, 2003). In the present study, refractory period was found to be very long, 2–4 h. Recovery from refractoriness was completely inhibited by inhibitors of eukaryote-type protein synthesis, cycloheximide or anisomysin, but not by inhibitors of prokaryote-type protein synthesis. This suggested that proteinous factor(s) responsible for generation of the depolarization is lost or inactivated upon depolarization and synthesized during the resting state. Low temperature, which is supposed to inhibit protein synthesis, also inhibited recovery from refractoriness. When unstimulated internodal cells were incubated in the medium containing an inhibitor of eukaryote-type protein synthesis, generation of the depolarization was almost completely inhibited. This result suggested that the factor is slowly turning over even in the absence of osmotic stimulation.     

Electrical perception of "death message" in chara: analysis of rapid component and ionic process

Electrical response upon wounding was analyzed in Chara corallina. A specimen comprising two adjoining internodal cells was prepared. One cell (victim cell) was killed by cutting and any changes in the membrane potential of the neighboring cell (the receptor cell) were measured. Upon cutting the victim cell, the receptor cell generated four kinds of depolarizations: (1) rapid component, (2) slow and long-lasting component, (3) action potential and (4) small spike. Rapid and slow components were observed in most cells. On the other hand, the action potential and small spike were not always ubiquitous among specimens. When an action potential was generated just after cutting the victim cell, the rapid component could not be observed due to masking by the action potential. It was suggested that both rapid and slow components were generated at the nodal end. On the other hand, action potentials were thought to be generated at the flank of the receptor cell. High turgor pressure of the cell was necessary for generating both rapid and slow components. Experiments under K(+)-induced depolarization unequivocally showed that the Cl(-) channel at the nodal end of the receptor cell was activated upon cutting the victim cell.     

After-Potentials and Large Depolarizations of Single Nodes of Ranvier Treated with Veratridine

Potential differences between normal nodes of Ranvier (single fiber from the sciatic nerve of the frog, air-gap method) and a node exposed to 1 to 2.5 x 10^-6 gm veratridine per ml were measured. Negative after-potentials occurred immediately after application of the alkaloid when spike configuration and resting potential were virtually unchanged. The after-potentials decreased in magnitude and their time constant increased as the resting membrane was depolarized either by outward currents or by a train of impulses. Increase of (Na)_o markedly increased the amplitude of the after-potential. After prolonged application of veratridine or with higher concentrations, a large slow depolarization (rate of potential change about 7 mv per second) could be triggered by a train of impulses or even a single spike. This depolarization could promptly be terminated by withdrawing Na. It is concluded that, once the nodal membrane has become permeable to Na (as during a spike), veratridine prevents the normal return of P_Na to its resting value.     

ANATOMY OF NODES VS. INTERNODES IN COLEUS: THE NODAL CAMBIUM

Secondary growth begins in the nodal regions before the internodal regions in Coleus, so that longitudinally discontinuous vascular cambia are formed in the 6th through the 9th or 10th nodes, where the internodal cambium becomes continuous between nodal cambia. The nodal cambia are identifiable by radial seriation in interfascicular regions, typical cytology of fusiform initials, and the presence of a ray system. Anatomical features distinct from the primary plant body are shared by the nodal and internodal cambia. Branching of primary vascular strands, restricted to procambium and phloem, is virtually confined to nodal regions. In secondary growth, vascular branching of xylem and phloem occurs in both nodes and internodes. Xylem strand branches are formed only from derivatives of vascular cambia. It is proposed that the cambium provides the secondary plant body an efficient channel for lateral auxin transport, by which branching across interfascicular regions is facilitated.     

The fine structure of the conducting system of the monkey heart (Macaca mulatta)

Summary In the sino-atrial (S-A) node of the monkey heart two types of muscle cells occur: 1. typical nodal cells which are the predominant cells and form the nodal fibers. 2. Intercalated clear cells with various diameters (4 to 12 m) and containing poorly developed myofibrils, rich in glycogen and demonstrating poor staining properties. These latter cells are dispersed, few in number, and never form discrete fibers of themselves, but are intercalated between the cell rows of the typical nodal fibers. Such intercalated clear cells become more numerous at the periphery of the node. Interconnection between the S-A node and the conventional atrial muscle is established by a progressive transformation of nodal fibers into atrial fibers producing an intermediate (or junctional) type of fiber at the nodal periphery. However, in addition, few nodal fibers make direct contact with the atrial cardiocytes. Our light and EM studies have failed to prove the existence of truly specialized internodal pathways. Nevertheless intercalated clear cells, nodal-like cells, junctional or intermediate type of cells are relatively frequent in valvular regions (Thebesian, Eustachian, A-V, fossa ovalis) and less frequent in other regions of the atrial wall.This study was conducted in part in the Department of Histology and Embryology of the Medical University in Budapest.     

Role of calcium ion in the excitability and electrogenic pump activity of theChara corallina membrane: I. Effects of La3+, verapamil,EGTA, W-7, and TFP on the action potential

Summary The cytoplasmic streaming of the normal internodal cell of giant algaChara stops transiently at about the peak of action potential. Application of La³⁺ or verapamil (a calcium channel blocker) or removal of external Ca²⁺ by EGTA caused a partial depolarization of the resting potential, partial decrease of the membrane conductance and a marked decrease of the amplitude of action potential. Under these conditions, the conductance at the peak of action potential reduced markedly and the streaming of cytoplasm did not cease during action potential (excitation-cessation (EC) uncoupling). The effects of Ca²⁺ channel blockers could not be removed by addition of CaCl₂ to the external medium. In contrast, the effect of EGTA on the excitability could be removed to a greater extent and the cytoplasmic streaming ceased at about the peak of action potential by the addition of Ca²⁺ externally. Application of calmodulin antagonists W-7 or TFP caused similar effects on the action potential and on the cytoplasmic streaming.